Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94.
OBJECTIVE
To describe mRNA for Lp82, a lens-specific form of calcium-activated protease (EC 34.22.17, calpain) in rat.


METHODS
Total RNA was extracted from lenses of 12-day-old rats, reverse transcribed, and amplified using polymerase chain reaction (PCR) with degenerate primers for the active site of calpains. Tissue specificity and relative amounts of mRNA for Lp82 in rat lens and other tissues were demonstrated by reverse transcription-PCR using gene-specific primers. PCR products were subcloned and sequenced using standard techniques.


RESULTS
Abundant amounts of mRNA for Lp82, a variant of muscle-type calpain p94, were discovered only in lens and not in seven other tissues assayed. When translated, the Lp82 mRNA would code for protein containing the usual cysteine active site residues and calcium-binding domains. Remarkably, the Lp82 cDNA product was identical to that of muscle-type calpain p94, except for deletions of most of the nucleotides coding for three unique insert regions termed NS, IS1, and IS2 found in p94. Compared with p94, a different exon 1 was present, and the nucleotides for all exons 6, 15, and 16 were deleted in Lp82.


CONCLUSIONS
Lp82 is a lens-specific calpain. It is a splice variant of muscle-type calpain p94, resulting from deletions of IS1 and IS2 regions and a different exon 1, possibly from an alternative, lens-specific promoter in the p94 gene. Lp82 is important because it may have lens-specific functions suited to the unique properties of the lens. Lp82 may also give insight into the functions of NS, IS1, and IS2 regions in p94, which is important in normal muscle physiology and in the study of limb-girdle muscular dystrophy type 2A in humans.
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Department of Oral Molecular Biology, Oregon Health Sciences University, Portland 97201, USA.
Fukiage
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Azuma
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Shearer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
D017931:DNA Primers; D004274:DNA, Recombinant; D012333:RNA, Messenger; D012313:RNA; D002154:Calpain; C078134:calpain p94